Blaesi, E. J., Palowitch, G. M., Hu, K., Kim, A. J., Rose, H. R., Alapati, R. B., Lougee, M. G., Kim, H. J., Taguchi, A. T., Tan, K. O., Laremore, T. N., Griffin, R. G., Krebs, C., Matthews, M. L., Silakov, A., Bollinger, M. J. Jr., Allen, B. D., Boal, A. K. Metal-free class I ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical. PNAS, September, 2018.
All cells obtain 2′-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, β; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in β. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the β is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.
Taguchi, A. T., Ohmori, D., Dikanov, S. A., and Iwasaki, T. g-Tensor Directions in the Protein Structural Frame of Hyperthermophilic Archaeal Reduced Rieske-Type Ferredoxin Explored by 13C Pulsed Electron Paramagnetic Resonance. Biochemistry, Vol. 57, No. 28, pp. 4074-4082, June, 2018.
Interpretation of magnetic resonance data in the context of structural and chemical biology requires prior knowledge of the g-tensor directions for paramagnetic metallo-cofactors with respect to the protein structural frame. Access to this information is often limited by the strict requirement of suitable protein crystals for single-crystal electron paramagnetic resonance (EPR) measurements or the reliance on protons (with ambiguous locations in crystal structures) near the paramagnetic metal site. Here we develop a novel pulsed EPR approach with selective 13Cβ-cysteine labeling of model [2Fe-2S] proteins to help bypass these problems. Analysis of the 13Cβ-cysteine hyperfine tensors reproduces the g-tensor of the Pseudomonas putida ISC-like [2Fe-2S] ferredoxin (FdxB). Its application to the hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus, for which the single-crystal EPR approach was not feasible, supports the best-fit gx-, gz-, and gy-tensor directions of the reduced cluster as nearly along Fe–Fe, S–S, and the cluster plane normal, respectively. These approximate principal directions of the reduced ARF g-tensor, explored by 13C pulsed EPR, are less skewed from the cluster molecular axes and are largely consistent with those previously determined by single-crystal EPR for the cytochrome bc1-associated, reduced Rieske [2Fe-2S] center. This suggests the approximate g-tensor directions are conserved across the phylogenetically and functionally divergent Rieske-type [2Fe-2S] proteins.
Dikanov, S. A. and Taguchi, A. T. Two-dimensional Pulsed EPR Resolves Hyperfine Coupling Strain in Nitrogen Hydrogen Bond Donors of Semiquinone Intermediates. J. Phys. Chem. B., Vol. 122, No. 20, pp. 5205-5211, April, 2018.
Hydrogen bonding between semiquinone (SQ) intermediates and side-chain or backbone nitrogens in protein quinone processing sites (Q-sites) is a common motif. Previous studies on SQs from multiple protein environments have reported specific features in the 15N HYSCORE spectra not reproducible by a theory based on fixed hyperfine parameters, and the source of these lineshape distortions remained unknown. In this work, using the spectra of the SQ in the Q-sites of wild-type and mutant D75H cytochrome bo3 ubiquinol oxidase from Escherichia coli, we have explained the observed additional features as originating from a-strain of the isotropic hyperfine coupling. In two-dimensional spectra, the a-strain manifests as well-resolved lineshape distortions of the basic cross-ridges and accompanying lines of low intensity in the opposite quadrant that allow its direct analysis. We have shown that their appearance is regulated by the relative values of the strain width, Δa, and parameter, δ = |2a + T| – 4ν15N. α-strain provides a direct measure of the structural dynamics and heterogeneity of the O···H···N bond in the SQ systems.
Taguchi, A. T., Miyajima-Nakano, Y., Fukazawa, R., Lin, M. T., Baldansuren, A., Gennis, R. B., Hasegawa, K., Kumasaka, T., Dikanov, S. A., and Iwasaki, T. Unpaired Electron Spin Density Distribution across Reduced [2Fe-2S] Cluster Ligands by 13Cβ-Cysteine Labeling. Inorg. Chem., Vol. 57, No. 2, pp. 741-746, December, 2017.
Iron–sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)n(Cys)4–n (n = 0, 1, 2) cluster by selective cysteine-13Cβ isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe2+ ligands than the nonreducible Fe3+ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe2+ ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution of all cysteine-13Cβ hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal–thiolate bonds not readily available to other techniques.
Greene, B. L., Taguchi, A. T., Stubbe, J., and Nocera, D. G. Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase. J. Am. Chem. Soc., Vol. 139, No. 46, pp. 16657-16665, October, 2017.
Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the E. coli class 1a RNR the thiyl radical (C439•) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α2:β2 subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y122β ↔ [W48β?] ↔ Y356β ↔ Y731α ↔ Y730α ↔ C439α). The mutant R411A(α) disrupts the H-bonding environment and conformation of Y731, ostensibly breaking the RT pathway in α2. However, the R411A protein retains significant enzymatic activity, suggesting Y731 is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH2Y) by amber codon suppression at positions Y731 or Y730 and investigation of the NH2Y• trapped state in the active α2:β2 complex by HYSCORE spectroscopy validate that the perturbed conformation of Y731 in R411A-α2 is dynamic, reforming the H-bond between Y731 and Y730 to allow RT to propagate to Y730. Kinetic studies facilitated by photochemical radical generation reveal that Y731 changes conformation on the ns−μs time scale, significantly faster than the enzymatic kcat. Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 × 104 s–1 when comparing wild type to R411A-α2, respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.
Taguchi, A. T., O’Malley, P. J., Wraight, C. A., and Dikanov, S. A. Determination of the Complete Spin Density Distribution in 13C-Labeled Protein-Bound Radical Intermediates Using Advanced 2D Electron Paramagnetic Resonance Spectroscopy and Density Functional Theory. J. Phys. Chem. B, Vol. 121, No. 44, pp. 10256-10268, October, 2017. (4 minute LiveSlides presentation narrated by Taguchi, A. T. available at http://pubs.acs.org/doi/suppl/10.1021/acs.jpcb.7b10036)
Determining the complete electron spin density distribution for protein-bound radicals, even with advanced pulsed electron paramagnetic resonance (EPR) methods, is a formidable task. Here we present a strategy to overcome this problem combining multifrequency HYSCORE and ENDOR measurements on site-specifically 13C-labeled samples with DFT calculations on model systems. As a demonstration of this approach, pulsed EPR experiments are performed on the primary QA and secondary QB ubisemiquinones of the photosynthetic reaction center from Rhodobacter sphaeroides 13C-labeled at the ring and tail positions. Despite the large number of nuclei interacting with the unpaired electron in these samples, two-dimensional X- and Q-band HYSCORE and orientation selective Q-band ENDOR resolve and allow for a characterization of the eight expected 13C resonances from significantly different hyperfine tensors for both semiquinones. From these results we construct, for the first time, the most complete experimentally determined maps of the s- and pπ-orbital spin density distributions for any protein organic cofactor radical to date. This work lays a foundation for understanding the relationship between the electronic structure of semiquinones and their functional properties, and introduces new techniques for mapping out the spin density distribution that are readily applicable to other systems.
Lin, Q.,* Parker, M. J.,* Taguchi, A. T.,* Ravichandran, K.,* Kim, A., Kang, G., Shao, J., Drennan, C. L., and Stubbe, J. Glutamate 52-β at the α/β Subunit Interface of E. coli Class Ia Ribonucleotide Reductase is essential for Conformational Gating of Radical Transfer. J. Biol. Chem., Vol. 292, No. 22, pp. 9229-9239, April, 2017. (*authors contributed equally to this paper)
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/β) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2β2 created from X-ray structures of α and β and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the α/β interface (Glu350 and Glu52 in β2 and Arg329 and Arg639 in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-β2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (F3Y122•) replacing the stable Tyr122• in WT-β2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His6-α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr356•-β2), 0.5 eq of dCDP/F3Y122•, and formation of an α2β2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2β2 complex formed.
Ravichandran, K. R., Zong, A. B., Taguchi, A. T., Nocera, D. G., Stubbe, J., and Tommos, C. Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer. J. Am. Chem. Soc., Vol. 139, No. 8, pp. 2994-3004, February, 2017.
Redox-active tyrosines (Ys) play essential roles in enzymes involved in primary metabolism including energy transduction and deoxynucleotide production catalyzed by ribonucleotide reductases (RNRs). Thermodynamic characterization of Ys in solution and in proteins remains a challenge due to the high reduction potentials involved and the reactive nature of the radical state. The structurally characterized α3Y model protein has allowed the first determination of formal reduction potentials (E°′) for a Y residing within a protein (Berry, B. W.; Martı́nez-Rivera, M. C.; Tommos, C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 9739–9743). Using Schultz’s technology, a series of fluorotyrosines (FnY, n = 2 or 3) was site-specifically incorporated into α3Y. The global protein properties of the resulting α3(3,5)F2Y, α3(2,3,5)F3Y, α3(2,3)F2Y and α3(2,3,6)F3Y variants are essentially identical to those of α3Y. A protein film square-wave voltammetry approach was developed to successfully obtain reversible voltammograms and E°’s of the very high-potential α3FnY proteins. E°′(pH 5.5; α3FnY(O•/OH)) spans a range of 1040 ± 3 mV to 1200 ± 3 mV versus the normal hydrogen electrode. This is comparable to the potentials of the most oxidizing redox cofactors in nature. The FnY analogues, and the ability to site-specifically incorporate them into any protein of interest, provide new tools for mechanistic studies on redox-active Ys in proteins and on functional and aberrant hole-transfer reactions in metallo-enzymes. The former application is illustrated here by using the determined α3FnY ΔE°’s to model the thermodynamics of radical-transfer reactions in FnY-RNRs and to experimentally test and support the key prediction made.
Ravichandran, K., Minnihan E. C., Lin Q., Yokoyama K., Taguchi A. T., Shao J., Nocera, D. G., and Stubbe, J. Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase. Biochemistry, Vol. 56, pp. 856-868, January, 2017.
Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2β2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in β2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2) across the α/β interface. The absence of an α2β2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in β2 suggested its importance in catalysis and/or conformational gating. To study its function, pH–rate profiles of wild-type β2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating, the strong oxidants, NO2Y122•-β2 and 2,3,5-F3Y122•-β2, were reacted with α2, CDP, and ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.
Taguchi, A. T., Baldansuren, A., and Dikanov, S. A. Basic and Combination Cross-Features in X- and Q-band HYSCORE of the 15N Labeled Bacteriochlorophyll a Cation Radical. Zeitschrift für Physikalische Chemie, Vol. 231, pp. 725-744, February, 2017.
Chlorophylls are an essential class of cofactors found in all photosynthetic organisms. Upon absorbing a photon, the excited state energy of the chlorophyll can either be transferred to another acceptor molecule, or be used to drive electron transfer. When acting as the primary donor in the bacterial photosynthetic reaction center, light-induced charge separation results in the formation of a cationic bacteriochlorophyll dimer. The hyperfine interactions between the unpaired electron of the 15N labeled bacteriochlorophyll cation radical and its four pyrrole nitrogens are probed with X- and Q-band 15N HYSCORE spectroscopy in frozen solution. The powder-type HYSCORE shows the basic (να(β), νβ(α)) cross-features as well as several types of combination cross-features. The nitrogen tensors were resolved in the squared-frequency representation of the HYSCORE spectra, and simulations of the combination peaks allowed for further refinement of the hyperfine coupling constants. The nitrogen tensors were found to have coupling constants a=3.28 MHz, T=1.23 MHz (N1 and N2), a=4.10 MHz, T=1.25 MHz (N3), and a=4.35 MHz, T=1.70 MHz (N4). The combination features were assigned based on a linear regression analysis of the cross-ridges in the squared-frequency representation as well as spectral simulations. The methodology discussed here will provide an important foundation for analyzing and understanding complex two-dimensional spectra from severalI=1/2 nuclei.
Sun, C., Taguchi, A. T., Vermaas, J. V., Beal, N. J., O’Malley, P. J., Tajkhorshid, E., Gennis, R. B., Dikanov, S. A. Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochrome bo3 from Escherichia coli. Biochemistry, Vol. 55, pp. 5714-5725, September, 2016.
The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron–nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz′ = 11.8 MHz, whereas for H2, Tz′ = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo3 QH site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.
Ravichandran, K. R., Taguchi, A. T., Wei, Y., Tommos, C., Nocera, D. G., Stubbe, J. A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes. JACS, Vol. 138, No. 41, pp 13706–13716, September, 2016.
Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y122·) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2). Equilibration of Y356·, Y731·, and Y730· was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y122· and Y356· has not been detected. Our recent report of Y356· formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y122 (F3Y122·-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F3Y122· and Y356·. Incubation of F3Y122·-β2, Y731F-α2 (or Y730F-α2), CDP, and ATP at different temperatures (2 − 37 °C) provides ΔE°′(F3Y122· − Y356·) of 20 ± 10 mV at 25 °C. The pH dependence of the F3Y122· ⇆ Y356· interconversion (pH 6.8 −8.0) reveals that the proton from Y356 is in rapid exchange with solvent, in contrast to the proton from Y122. Insertion of 3,5-difluorotyrosine (F2Y) at Y356 and rapid freeze-quench EPR analysis of its reaction with Y731F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F2Y356· generation by the native Y122·.
Sun, C., Taguchi, A. T., Beal, N., O’Malley, P. J., Dikanov, S. A., and Wraight, C. A. Regulation of the Primary Quinone Binding Conformation by the H Subunit in Reaction Centers from Rhodobacter sphaeroides. J. Phys. Chem. Lett., Vol. 6, No. 22, pp. 4541-4546, October, 2015.
Unlike photosystem II (PSII) in higher plants, bacterial photosynthetic reaction centers (bRCs) from Proteobacteria have an additional peripheral membrane subunit “H”. The H subunit is necessary for photosynthetic growth, but can be removed chemically in vitro. The remaining LM dimer retains its activity to perform light-induced charge separation. Here we investigate the influence of the H subunit on interactions between the primary semiquinone and the protein matrix, using a combination of site-specific isotope labeling, pulsed electron paramagnetic resonance (EPR), and density functional theory (DFT) calculations. The data reveal substantially weaker binding interactions between the primary semiquinone and the LM dimer than observed for the intact bRC; the amount of electron spin transferred to the nitrogen hydrogen bond donors is significantly reduced, the methoxy groups are more free to rotate, and the spectra indicate a heterogeneous mixture of bound semiquinone states. These results are consistent with a loosening of the primary quinone binding pocket in the absence of the H subunit.
Yi, S., Taguchi, A. T., Samoilova, R. I., O’Malley, P. J., Gennis, R. B., and Dikanov, S. A. Plasticity in the High Affinity Menaquinone Binding Site of the Cytochrome aa3-600 Menaquinol Oxidase from Bacillus subtilis. Biochemistry, Vol. 54, No. 32, pp. 5030-5044, July, 2015.
Cytochrome aa3-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochrome c when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made the R70H mutant and have characterized the menasemiquinone radical by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a14N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e2qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa3-600, the SQ is instead hydrogen bonded with Nε from the Arg70 side chain. Analysis of the 1H 2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function.
Taguchi, A. T., O’Malley, P. J., Wraight, C. A., and Dikanov, S. A. Hydrogen Bond Network around the Semiquinone of the Secondary Quinone Acceptor QB in Bacterial Photosynthetic Reaction Centers. J. Phys. Chem. B, Vol. 119, No. 18, pp. 5805-5814, April, 2015.
By utilizing a combined pulsed EPR and DFT approach, the high-resolution structure of the QB site semiquinone (SQB) was determined. The development of such a technique is crucial toward an understanding of protein-bound semiquinones on the structural level, as (i) membrane protein crystallography typically results in low resolution structures, and (ii) obtaining protein crystals in the semiquinone form is rarely feasible. The SQB hydrogen bond network was investigated with Q- (∼34 GHz) and X-band (∼9.7 GHz) pulsed EPR spectroscopy on fully deuterated reactions centers from Rhodobacter sphaeroides. Simulations in the SQB g-tensor reference frame provided the principal values and directions of the H-bond proton hyperfine tensors. Three protons were detected, one with an anisotropic tensor component, T = 4.6 MHz, assigned to the histidine NδH of His-L190, and two others with similar anisotropic constants T = 3.2 and 3.0 MHz assigned to the peptide NpH of Gly-L225 and Ile-L224, respectively. Despite the strong similarity in the peptide couplings, all hyperfine tensors were resolved in the Q-band ENDOR spectra. The Euler angles describing the series of rotations that bring the hyperfine tensors into the SQB g-tensor reference frame were obtained by least-squares fitting of the spectral simulations to the ENDOR data. These Euler angles show the locations of the hydrogen bonded protons with respect to the semiquinone. Our geometry optimized model of SQB used in previous DFT work is in strong agreement with the angular constraints from the spectral simulations, providing the foundation for future joint pulsed EPR and DFT semiquinone structural determinations in other proteins.
Vermaas, J. V., Taguchi, A. T., Dikanov, S. A., Wraight, C. A., and Tajkhorshid, E. Redox Potential Tuning through Differential Quinone Binding in the Photosynthetic Reaction Center of Rhodobacter sphaeroides. Biochemistry, Vol. 54, No. 12, pp. 2104-2116, March, 2015.
Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force field and subsequent molecular dynamics simulations of the quinone-bound RC, we have investigated and characterized the interactions of the protein with the quinones in the QA and QB sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the QB site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential difference between the quinones occupying the QA and QB sites. Disruption of this interaction leads to weaker binding in a ubiquinone analogue that lacks a 2-methoxy group, a finding supported by reverse electron transfer electron paramagnetic resonance experiments of the QA–QB– biradical and competitive binding assays.
Hong, S., De Almeida, W., Taguchi, A. T., Samoilova, R. I., Gennis, R. B., O’Malley P. J., Dikanov, S. A., and Crofts, A. R. The Semiquinone at the Qi Site of the bc1 Complex Explored Using HYSCORE Spectroscopy and Specific Isotopic Labeling of Ubiquinone in Rhodobacter sphaeroides via 13C Methionine and Construction of a Methionine Auxotroph. Biochemistry, Vol. 53, No. 38, pp. 6022-6031, September, 2014.
Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with L-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly.
Samoilova, R. I., Taguchi, A. T., O’Malley P. J., Dikanov, S. A., and Lugtenburg, J. Hyperfine Interaction Tensors of 13C Nuclei for Ring Carbons of Ubisemiquinone-10 Hydrogen Bonded in Alcohol Solvents. Appl. Magn. Reson., Vol. 45, No. 9, pp. 941-953, September, 2014.
The anion radicals of ubiquinones-10 13C chemically labeled at the C5 or C6 ring positions in alcohol have been studied by 1D and 2D ESEEM to define the hyperfine interaction tensors with the 13C nuclei. Analysis of the cross-peak line shapes and simulations of the spectra allowed us to conclude that the hyperfine tensors are characterized by an anisotropic component T ~6 MHz and an isotropic coupling a ~−3 MHz with support from DFT calculations. However, these values were found to be inconsistent with the shift of the sum combination harmonic in the four-pulse ESEEM spectra. Simulations resolve this apparent discrepancy by showing that the shift of the sum combination to lower frequency and its broadening can be accounted for by a distribution of the hyperfine couplings. A spread of the methoxy group conformations, as supported by previous experimental observations, is suggested as the mechanism influencing the distribution of the hyperfine couplings for the ring carbons.
Taguchi, A. T., O’Malley, P. J., Wraight, C. A., and Dikanov, S. A. Hyperfine and Nuclear Quadrupole Tensors of Nitrogen Donors in the QA Site of Bacterial Reaction Centers: Correlation of the Histidine Nδ Tensors with Hydrogen Bond Strength. J. Phys. Chem. B, Vol. 118, No. 31, pp. 9225-9237, July, 2014.
X- and Q-band pulsed EPR spectroscopy was applied to study the interaction of the QA site semiquinone (SQA) with nitrogens from the local protein environment in natural abundance 14N and in 15N uniformly labeled photosynthetic reaction centers of Rhodobacter sphaeroides. The hyperfine and nuclear quadrupole tensors for His-M219 Nδ and Ala-M260 peptide nitrogen (Np) were estimated through simultaneous simulation of the Q-band 15N Davies ENDOR, X- and Q-band 14,15N HYSCORE, and X-band 14N three-pulse ESEEM spectra, with support from DFT calculations. The hyperfine coupling constants were found to be a(14N) = 2.3 MHz, T = 0.3 MHz for His-M219 Nδ and a(14N) = 2.6 MHz, T = 0.3 MHz for Ala-M260 Np. Despite that His-M219 Nδ is established as the stronger of the two H-bond donors, Ala-M260 Np is found to have the larger value of a(14N). The nuclear quadrupole coupling constants were estimated as e2Qq/4h = 0.38 MHz, η = 0.97 and e2Qq/4h = 0.74 MHz, η = 0.59 for His-M219 Nδ and Ala-M260 Np, respectively. An analysis of the available data on nuclear quadrupole tensors for imidazole nitrogens found in semiquinone-binding proteins and copper complexes reveals these systems share similar electron occupancies of the protonated nitrogen orbitals. By applying the Townes–Dailey model, developed previously for copper complexes, to the semiquinones, we find the asymmetry parameter η to be a sensitive probe of the histidine Nδ–semiquinone hydrogen bond strength. This is supported by a strong correlation observed between η and the isotropic coupling constant a(14N) and is consistent with previous computational works and our own semiquinone-histidine model calculations. The empirical relationship presented here for a(14N) and η will provide an important structural characterization tool in future studies of semiquinone-binding proteins.
De Almeida, W., Taguchi, A. T., Dikanov, S. A., Wraight, C. A., and O’Malley, P. J. The 2-Methoxy Group Orientation Regulates the Redox Potential Difference between the Primary (QA) and Secondary (QB) Quinones of Type II Bacterial Photosynthetic Reaction Centers. J. Phys. Chem. Lett., Vol. 5, No. 15, pp. 2506-2509, June, 2014. (5 minute LiveSlides presentation narrated by Taguchi, A. T. available here)
Recent studies have shown that only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from purple bacteria such as Rb. sphaeroides. 13C HYSCORE measurements of the 2-methoxy group in the semiquinone states, SQA and SQB, were compared with DFT calculations of the 13C hyperfine couplings as a function of the 2-methoxy dihedral angle. X-ray structure comparisons support 2-methoxy dihedral angle assignments corresponding to a redox potential gap (ΔEm) between QA and QB of 175–193 mV. A model having a methyl group substituted for the 2-methoxy group exhibits no electron affinity difference. This is consistent with the failure of a 2-methyl ubiquinone analogue to function as QB in mutant reaction centers with a ΔEm of ∼160–195 mV. The conclusion reached is that the 2-methoxy group is the principal determinant of electron transfer from QA to QB in type II photosynthetic reaction centers with ubiquinone serving as both acceptor quinones.
Taguchi, A. T., O’Malley, P. J., Wraight, C. A., and Dikanov, S. A. Nuclear hyperfine and quadrupole tensor characterization of the nitrogen hydrogen bond donors to the semiquinone of the QB site in bacterial reaction centers: A combined X- and S-band 14,15N ESEEM and DFT study. J. Phys. Chem. B, Vol. 118, No. 6, pp. 1501-1509, February, 2014.
The secondary quinone anion radical QB– (SQB) in reaction centers of Rhodobacter sphaeroidesinteracts with Nδ of His-L190 and Np (peptide nitrogen) of Gly-L225 involved in hydrogen bonds to the QB carbonyls. In this work, S-band (∼3.6 GHz) ESEEM was used with the aim of obtaining a complete characterization of the nuclear quadrupole interaction (nqi) tensors for both nitrogens by approaching the cancelation condition between the isotropic hyperfine coupling and 14N Zeeman frequency at lower microwave frequencies than traditional X-band (9.5 GHz). By performing measurements at S-band, we found a dominating contribution of Nδ in the form of a zero-field nqi triplet at 0.55, 0.92, and 1.47 MHz, defining the quadrupole coupling constant K = e2qQ/4h = 0.4 MHz and associated asymmetry parameter η = 0.69. Estimates of the hyperfine interaction (hfi) tensors for Nδ and Np were obtained from simulations of 1D and 2D 14,15N X-band and three-pulse 14N S-band spectra with all nuclear tensors defined in the SQB g-tensor coordinate system. From simulations, we conclude that the contribution of Np to the S-band spectrum is suppressed by its strong nqi and weak isotropic hfi comparable to the level of hyperfine anisotropy, despite the near-cancelation condition for Np at S-band. The excellent agreement between our EPR simulations and DFT calculations of the nitrogen hfi and nqi tensors to SQB is promising for the future application of powder ESEEM to full tensor characterizations.
Taguchi, A. T., Mattis, A. J., O’Malley, P. J., Dikanov, S. A., and Wraight, C. A. Tuning Cofactor Redox Potentials: The 2-Methoxy Dihedral Angle Generates a Redox Potential Difference of >160 mV between the Primary (QA) and Secondary (QB) Quinones of the Bacterial Photosynthetic Reaction Center. Biochemistry, Vol. 52, No. 41, pp. 7164-7166, September, 2013.
Only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from Rhodobacter sphaeroides. 13C hyperfine sublevel correlation measurements of the 2-methoxy in the semiquinone states, SQAand SQB, were compared with quantum mechanics calculations of the 13C couplings as a function of the dihedral angle. X-ray structures support dihedral angle assignments corresponding to a redox potential gap (ΔEm) between QA and QB of ∼180 mV. This is consistent with the failure of a ubiquinone analogue lacking the 2-methoxy to function as QB in mutant reaction centers with a ΔEm of ≈160–195 mV.
Taguchi, A. T., O’Malley, P. J., Wraight, C. A., and Dikanov, S. A. Conformational Differences between the Methoxy Groups of QA and QB Site Ubisemiquinones in Bacterial Reaction Centers: A Key Role for Methoxy Group Orientation in Modulating Ubiquinone Redox Potential. Biochemistry, Vol. 52, No. 27, pp. 4648-4655, June, 2013.
Ubiquinone is an almost universal, membrane-associated redox mediator. Its ability to accept either one or two electrons allows it to function in critical roles in biological electron transport. The redox properties of ubiquinone in vivo are determined by its environment in the binding sites of proteins and by the dihedral angle of each methoxy group relative to the ring plane. This is an attribute unique to ubiquinone among natural quinones and could account for its widespread function with many different redox complexes. In this work, we use the photosynthetic reaction center as a model system for understanding the role of methoxy conformations in determining the redox potential of the ubiquinone/semiquinone couple. Despite the abundance of X-ray crystal structures for the reaction center, quinone site resolution has thus far been too low to provide a reliable measure of the methoxy dihedral angles of the primary and secondary quinones, QA and QB. We performed 2D ESEEM (HYSCORE) on isolated reaction centers with ubiquinones 13C-labeled at the headgroup methyl and methoxy substituents, and have measured the 13C isotropic and anisotropic components of the hyperfine tensors. Hyperfine couplings were compared to those derived by DFT calculations as a function of methoxy torsional angle allowing estimation of the methoxy dihedral angles for the semiquinones in the QA and QB sites. Based on this analysis, the orientation of the 2-methoxy groups are distinct in the two sites, with QB more out of plane by 20–25°. This corresponds to an ≈50 meV larger electron affinity for the QB quinone, indicating a substantial contribution to the experimental difference in redox potentials (60–75 mV) of the two quinones. The methods developed here can be readily extended to ubiquinone-binding sites in other protein complexes.